Method for identifying an intestinal phenotype

ABSTRACT

The present invention relates to a method for identifying cells having a predisposition to develop an intestinal phenotype, wherein the cells are characterized by the loss of expression of the RUNX3 gene and the expression of one or more intestinal marker genes. In particular, the invention is directed to the identification of cells, which exhibit an intestinal phenotype representing a precursor of gastric cancer. Furthermore, the invention discloses a method for identifying a compound inhibiting the development of an intestinal phenotype in cells having a predisposition to develop an intestinal phenotype. Finally, the invention also relates to kits of parts for performing these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application makes reference to and claims priority of theprovisional application for a “Method For Identifying An IntestinalPhenotype” filed earlier in the US Patent and Trademark Office on Jun.1, 2005, and there duly assigned Ser. No. 60/686,243, the contents ofwhich is incorporated herein by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to a method for identifying cells having apredisposition to develop an intestinal phenotype, wherein the cells arecharacterized by the loss of expression of the RUNX3 gene and theexpression of one or more intestinal marker genes. In particular, theinvention is directed to the identification of cells, which exhibit anintestinal phenotype representing a precursor of gastric cancer.Furthermore, the invention provides a method for identifying a compoundinhibiting the development of an intestinal phenotype in cells having apredisposition to develop an intestinal phenotype. Finally, theinvention also relates to kits of parts for performing these methods.

BACKGROUND OF THE INVENTION

Gastric cancer is the second leading cause of cancer mortalityworldwide, accounting for more than 650 000 deaths annually. Sincegastric cancers are largely resistant to chemotherapy and radiotherapy,it is of high importance to detect the development of this neoplasm atan early stage. However, early detection of gastric cancer is uncommon.

A detection method presently used is taking x-rays (radiography) of theesophagus, the stomach, and the upper gastrointestinal tract.Administration of a barium solution, and possibly pumping air into thestomach, is carried out to assist in identifying tumors or otherabnormal areas. The likelihood of detecting tumors using this method arehowever believed to be below 50%. A further detection method isendoscopy, an examination of the esophagus and stomach using a thin,lighted fiber optic tube termed “gastroscope”. To confirm accuratediagnosis, the removal of the respective mucosal tissue by endoscopicresection is often required (for most recent data see e.g. Jang, B. K.et al. [2006] Gastrointestinal Endoscopy 63, 5, AB105, S1425), whichcoincides with cancer treatment. Other screening methods, such asradiographic fluorography or the determination of serum pepsinogenratios of PGI to PGII, but these are rather experimental and may notdetect gastric cancer in the early stages.

There are generally no symptoms in the early stages of gastric cancer,so that the cancer has in many cases spread before it is detected. Whensymptoms do occur, they are often so vague and nonspecific that patientsignore them. Current laboratory tests for tumor markers are of no value,unless there is already a metastatic liver spread (see e.g. Tierney etal., Current Medical Diagnosis & Treatment 2006, Lange/Mc Graw Hill,pages 596-599). Accordingly, detection of gastric cancer in the earlystages or at the stage of intestinal metaplasia may help make routinescreening easier facilitating early detection and treatment.

Colonization of the gastric mucosa by Helicobacter pylori, which isconsidered to confer a high risk for gastric cancer, is known to causechronic gastritis followed by atrophic gastritis and intestinalmetaplasia (INTESTINAL METAPLASIA). While the molecular eventsassociated with Helicobacter infection have been well studied in recentyears (Peek, R. M. and Blaser, M. J. (2002) Nat. Rev. Cancer 2, 28-37),the genetic and epigenetic changes required for the initiation ofgastric carcinogenesis are still poorly understood, partly because thegenes involved in the regulation of growth and differentiation of thestomach epithelium, as well as those involved in carcinogenesis, are notknown.

Gastric cancer can be histologically divided into a diffuse type and anintestinal type (Lauren, P. (1965) Acta Pathol. Microbiol. Scand. 64,31-49). Inactivation of the E-cadherin gene is frequently involved inthe diffuse type of familial cancer (Guilford, P. et al. (1998) Nature392, 402-405) as well as in sporadic cases (Yuasa, Y. (2003) Nat. Rev.Cancer 3, 592-600). Although genetic and epigenetic alterations in theAPC, MLH1, TP53, and TGF-β type 11 receptor genes, and overexpression ofthe erbB-2 and cyclin E genes have been observed in the intestinal aswell as diffuse types (Yuasa, Y. (2003), supra), they only occur in alimited number of cases and have no known roles at the initiation ofcarcinogenesis.

Recently, a causal relationship between the loss of expression of theRunt-related (RUNX) gene RUNX3 and gastric cancer has been identified(Li, Q. L. et al. (2002) Cell 109, 113-124; International PatentApplication WO 02/061069, which is incorporated by reference in itsentirety herein). The transcription factor subunit RUNX3, a mousehomolog of the product of the Drosophila segmentation gene runt, is amajor nuclear target of the TGF-β signaling pathway (Lund, A. H. and vanLohuizen, M. (2002) Cancer Cell 1, 213-215; Ito, Y. and Miyazono, K.(2003) Curr. Opin. Genet. Dev. 13, 43-47). RUNX3 mediates TGF-β-inducedgrowth inhibition and apoptosis of gastric epithelial cells. The humanRUNX3 gene can be inactivated by hemizygous deletion and silencing dueto hypermethylation of the promoter region in 40% of stage I and over90% of stage 1V gastric cancers, suggesting that inactivation of RUNX3takes place in the early stages of carcinogenesis as well as duringprogression. The tumorigenicity in nude mice of a gastric cancer cellline that failed to express RUNX3 was strongly inhibited by theexogenous expression of RUNX3, but this inhibitory activity was notobserved for a RUNX3 allele bearing a rare single amino acidsubstitution, R122C, which was identified in a gastric cancer patient.Furthermore, cell lines isolated from the gastric epithelium of RUNX3−/−mice were tumorigenic when injected into nude mice whereas thoseisolated from wild type mice were not. These results suggest that RUNX3is a tumor suppressor of gastric cancer (Li, Q. L. et al. (2002), supra;International Patent Application WO 02/061069).

With respect to the role of RUNX3 in carcinogenesis, it has beenobserved that RUNX3 expression is greatly reduced in INTESTINALMETAPLASIA, a tissue frequently observed in association with gastriccancer and characterized by the morphological changes of gastricepithelial cells into cells resembling intestinal epithelial cells(Stemmermann, G. N. (1994) Cancer 74, 556-564). Epidemiologically,INTESTINAL METAPLASIA has been shown to be closely associated withgastric cancer (Sugano, H. et al. (1986) GANN Monogr. Cancer Res. 31,53-58) and has been discussed as being indicative for the presence of apre-cancerous state. However, opposing views persist as to therelationship between a pre-cancerous state and INTESTINAL METAPLASIA,and the nature of a relationship, if any, between INTESTINAL METAPLASIAand gastric cancer has not been established.

Accordingly, it is an object of the invention to provide a method thatallows for the detection of a cell stage that corresponds to an earlyonset of gastric carcinogenesis.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for identifying one ormore cells having a predisposition to develop an intestinal phenotype,the method includes detecting in the one or more cells the loss ofexpression of the RUNX3 gene. The method also includes detecting in theone or more cells the expression of one or more intestinal marker genes.

The cells may for instance be isolated, purified and/or cultured. Thecells may also be included in a mammal, such as a human.

In some embodiments, when detecting in the one or more cells theexpression of one or more intestinal marker genes, also the expressionof one or more gastric marker genes is detected.

In some embodiments, the method further includes comparing the result ofthe measurements obtained in detecting the loss of expression of theRUNX3 gene and in detecting the expression of one or more intestinalmarker genes with those of obtained in a control cell.

In another aspect, the invention provides a method for identifying acompound that inhibits, or is capable of inhibiting, the development ofan intestinal phenotype in one or more cells that have a predispositionto develop an intestinal phenotype. The respective cells arecharacterized by the loss of expression of the RUNX3 gene and by theexpression of one or more intestinal marker genes. The method includes:

(a) contacting the one or more cells with a solution supposed to containat least one compound to be identified;

(b) incubating the cells for a predetermined period of time; and

(c) measuring the expression of the RUNX3 gene as well as the expressionof said one or more intestinal marker genes. (b) may also includedetecting the expression of one or more gastric marker genes

In some embodiments the method further includes:

(d) comparing the result of the measurement obtained in step (c) withthat of a control measurement without the addition of said solutionsupposed to contain said at least one compound to be identified; and

(e) identifying the compound inhibiting the development of saidintestinal phenotype.

In yet another aspect, the invention provides kits of parts forperforming the methods outlined above.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the drawings, in which:

FIG. 1 depicts a diagram of a gastric gland (a) and chief cells andsurface epithelial cells in the stomach of Runx3−/− mice, withimmunohistochemical detection of MUC5AC (surface epithelial cells) (b-e,boxed regions in FIG. 1 b and 1 c are enlarged in (d) and (e),respectively), pepsinogen (f-l, boxed regions in FIGS. 1 f and 1 g areenlarged in (h) and (i), (j-o) Immunohistochemical detection of chiefcells (j, k), mucous neck cells (l, m) and parietal cells (m, o) withanti-pepsinogen, anti-MUC6 and anti-H⁺/K⁺ ATPase antibodies,respectively. (j-o) represent serial sections. (p) depicts the averagenumbers of chief cells, mucous neck cells and parietal cells in thefundic gland of 6 months-old WT and RUNX3−/− mice. (b-o) shows fundicglands of 6 months-old mice. Counter staining was done with hematoxylin(b-o). Scale bars are equal to 50 μm (d, e, h-o) and 100 μm (b, c, f,g), respectively.

FIG. 2 depicts the cellular proliferation in the Lo and inhibition ofapoptosis in the Up of Runx3−/− gastric epithelium.5-bromo-2-deoxyuridine incorporation (a, b), PCNA immunostaining (c, d),and a DNA fragmentation detection assay (e, f) are shown.

FIG. 3 shows intestinalization of RUNX3−/− gastric epithelial cells.Immunohistochemical detection of CDX2 (fundic glands of 6 months-oldmice) (a, b); MUC6 immunostaining, followed by Alcian Blue staining (3months-old gastric epithelia at the fundic area) (c, d); Alcian Bluestaining (6 months-old gastric epithelia at the fundic area) (e, f,boxed regions in FIG. 3 f are enlarged in g and h); andimmunohistochemical detection of ITF (i, j), MUC2 (k, l), villin (m:wild-type, n: RUNX3−/− Up, o: RUNX3−/− Lo) (all fundic glands of 6months-old mice); and immunohistochemical detection of ITF (pyloricglands of 6 months-old mice) (p, q, boxed regions in FIGS. 3 p and 3 qare enlarged in r and s, respectively).

FIG. 4 shows, by immunostaining, cells with the capacity to proliferatefrom dysplasia in RUNX3−/− gastric epithelium, wherein an anti-MUC6immunoglobulin, followed by Alcian Blue staining (a, e, f,),immunodetection of MUC6-expressing cells (b), double staining usingAlcian Blue and an anti-CDX2 immunoglobulin (c), an anti-PCNAimmunoglobulin (d), and hematoxylin and eosin staining performed on aserial section from FIG. 4 f (g) were used.

FIG. 5 depicts a model of the role of Runx3 in gastric carcinogenesis.

FIG. 6 depicts immunohistochemistry staining of MUC2 (A), MUC5AC (B),MUC6 (C) and CDX2 (D) in intestinal metaplasia.

FIG. 7 depicts the immunohistochemistry staining of MUC2 (A), MUC5AC(B), MUC6 (C), and CDX2 (D) in gastric dysplasia.

FIG. 8 depicts immunohistochemistry staining of MUC2 (A), MUC5AC (B),RUNX3 (C), and CDX2 (D) in gastric dysplasia.

DETAILED DESCRIPTION

The invention is based on the surprising finding that the inactivationof the RUNX3 gene expression finally results in the induction ofINTESTINAL METAPLASIA in gastric epithelial cells. The term “metaplasia”is understood as a conversion from a fully differentiated cell type toanother. While metaplasia may in some cases be a means of regeneration,it may likewise in other cases be an abnormal replacement of cells ofone type by those of another type. Intestinal metaplasia is a conversionin morphology of gastric epithelial cells to intestinal cells. Duringthis event gastric mucosa is replaced by epithelium that resembles thesmall and large bowel mucosa. Intestinal metaplasia results from gastricstem cells being diverted from a proliferation into cells that arespecific for the stomach towards a proliferation into cells of theintestine, such as absorptive cells, goblet cells and Paneth cells.Since intestinal metaplasia is often associated with gastric cancer, itis usually considered as an indication of a potential risk in developingintestinal-type gastric cancer. Nevertheless, intestinal metaplasia ispresent in only about 20% of all gastric biopsies and it is known thatonly few of them progress to gastric cancer. Therefore there is aconflict of opinions among pathologists and doctors as to whetherintestinal metaplasia might be considered a precancerous state orwhether it is merely associated with gastric cancer without a directlink.

In view of the inventors' findings (including Osaki, M. et al. (2004),European Journal of Clinical Investigation 34, 605-612, incorporatedherein by reference in its entirety), and considering the potentialfunction of RUNX3 as a tumor suppressor gene, INTESTINAL METAPLASIA mustbe regarded as a pre-cancerous state, wherein the inactivation of RUNX 3appears to trigger carcinogenesis. Dysplasia, which has to be consideredas being one step closer to cancer, has also been found by theapplicants as being characterized by a significantly reduced expressionof RUNX3, when compared to normal gastric cells. In gastric cancer RUNX3is likewise frequently inactivated (Ito, K. et al. (2005) Cancer Res.65, 7743-7.750; Nakase, Y, et al. (2005) British Journal of Cancer 92,562-569, incorporated herein by reference in their entirety).Furthermore, the progression of INTESTINAL METAPLASIA is accompanied bythe expression of several intestine-specific genes in gastric epithelialcells. These genes may thus be used as marker genes, being indicativefor a predisposition of the affected cells to transform into anintestinal phenotype and provide a basis for the development of methodsfor identifying such cells in order to detect gastric cancer in an earlystage. Therefore, the present invention allows for instance fordiagnosis and subsequently carrying out methods of prevention ortreatment of gastric cancer, such as for example by chemotherapy,including the activation of RUNX3 (see international patent applicationsWO 2005/115388 and WO 2002/061069, which are incorporated in theirentirety by reference herein), at a stage where previously an individualwas typically not even aware of an increased risk of, or the developmentof a tumor.

In a first aspect the present invention provides a method that allowsfor the identification of one or more cells that have a predispositionto develop an intestinal phenotype.

The term “intestinal phenotype” refers to the outward appearance of acell or a tissue exhibiting characteristics of an intestinal cell ortissue. Beyond this morphological definition, “intestinal phenotype”also refers to the function of an intestinal cell or tissue. Examples ofsuch phenotypes may relate to cell size (enlargement or reduction) orcell shape, cell proliferation (increase in cell number), celldifferentiation (change in physiological state), cytotoxicity (celldeath), apoptosis (programmed cell death) or cell survival. Typically anintestinal phenotype refers to a condition characterized by a retainedcapacity of the cells to proliferate, i.e. the capability of the cellsto divide and to propagate. As an illustrative example, an intestinalphenotype may be a precursor of gastric cancer, which relates to anyphenotype being indicative for a pre-cancerous state, i.e. anintermediate state in the transformation of a normal cell into a tumorcell. In some embodiments the intestinal phenotype is INTESTINALMETAPLASIA, a tissue frequently observed in association with gastriccancer and characterized by the morphological changes of gastricepithelial cells into cells resembling intestinal epithelial cells(Stemmermann, G. N. (1994) Cancer 74, 556-564).

The term “cell” as used herein refers to any cell or type of cell thatcan be subjected to the inventive methods such as an endothelial cell,an epithelial cell, a blood cell, a fibroblast, a myocyte or a neuron.In some embodiments of the invention, the one or more cells areepithelial cells, such as gastric epithelial cells, for instancesecretory epithelial cells. Examples of gastric secretory epithelialcells include, but are not limited to, mucous cells, parietal cells(secreting hydrochloric acid), chief cells (secreting the proteolyticenzyme pepsin) and G cells (secreting the hormone gastrin). Mucouscells, the most abundant gastric epithelial cells, secrete abicarbonate-rich mucus, which coats and lubricates the gastric surface,and extend down into the glands as “mucous neck cells”. Thus, anillustrative example of gastric epithelial cells are gastric mucous neckcells.

The one or more cells may be derived from any mammalian species, e.g.mouse, rat, guinea pig, rabbit, goat, sheep, monkey or human.Accordingly, in some embodiments the cells are of human origin. In someembodiments the cells are in their normal physiological environment, forexample included in a mammal. In some embodiments the cell(s) is/areisolated or purified. The term “purified” is understood to be a relativeindication in comparison to the original environment of the cell,thereby representing an indication that the cell is relatively purerthan in the natural environment. It therefore includes, but does notonly refer to an absolute value in the sense of absolute purity fromother cells (such as a homogeneous cell population). The term “isolated”indicates that the cell or cells has/have been removed from its/theirnormal physiological environment. Thus, the cell or cells may beincluded in a tissue sample or in an aqueous solution, or placed in adifferent physiological environment. In some embodiments the cell is/thecells are cultured.

The terms “expression” or “gene expression” as used herein relate to theentirety of regulatory pathways converting the information encoded inthe nucleic acid sequence of a gene first into messenger RNA (mRNA) andthen to a protein. Accordingly, the expression of a gene comprises itstranscription into a primary hnRNA, the processing of this hnRNA into amature RNA and the translation of the mRNA sequence into thecorresponding amino acid sequence of the protein. In this context, it isalso noted that the term “gene product” refers not only to a protein,including e.g. a final protein (including a splice variant thereof)encoded by that gene and a respective precursor protein whereapplicable, but also to the respective mRNA, which may be regarded asthe “first gene product” during the course of gene expression.

The present method includes detecting the loss of expression of theRUNX3 gene in the one or more cells.

The terms “RUNX3 gene” or “RUNX3” as used herein generally refer to thehuman DNA- and protein sequences respectively, as described by Bae, S.C. et al. (Gene 159, 245-248 (1995)), whereof the gene has the GenBankaccession number BC013362, and the protein has the UniProtKB/Swiss-Protaccession number Q13761. However, it is also understood that “RUNX 3” isnot limited to the human gene and protein isoforms (e.g. GenBankaccession number NM_(—)001031680) but includes all functionallyequivalent mammalian RUNX3 isoforms such as those from mouse, rat, goator monkey, to name a few of the various isoforms. As a few illustrativeexamples of respective proteins shall serve the mouse protein ofSwissProt accession number Q64131, the rat protein of UniProtKB/TrEMBLaccession number Q91ZK1, the Mongolian jird protein of UniProtKB/TrEMBLaccession number Q2 MHJ6, the Clearnose skate protein ofUniProtKB/TrEMBL accession number Q6SZR4 and the Zebrafish protein ofUniProtKB/TrEMBL accession number Q9DEA0.

The term “detecting” refers to any method that can be used to detect thepresence of a nucleic acid (DNA and RNA) or a protein. These methodscomprise established standard procedures well known in the art (cf. e.g.Ausubel, F. M. et al. (2001) Current Protocols in Molecular Biology.Wiley & Sons, Hoboken, N.J.). Examples of such methods are RT-PCR, RNAseprotection assay, Northern analysis, Western analysis, ELISA,radioimmunoassay or fluorescence titration assay. The detection methodmay include an amplification of the signal caused by the nucleic acid orprotein, such as a polymerase chain reaction (PCR) or the use of thebiotin-streptavidin system, for example in form of a conjugation to animmunoglobulin. The detection method may for example include the use ofan immunoglobulin, which may be linked to an attached label, such as forinstance in Western analysis or ELISA. Where desired, an intracellularimmunoglobulin may be used for detection. Some or all of the steps ofdetection may be part of an automated detection system. Illustrativeexamples of such systems are automated real-time PCR platforms,automated nucleic acid isolation platforms, PCR product analyzers andreal-time detection systems. The detection may for instance rely onspectroscopical, photochemical, photometric, fluorometric, radiological,or enzymatic or thermodynamic means. An example of a spectroscopicaldetection method is fluorescence correlation spectroscopy. Aphotochemical method is for instance photochemical cross-linking. Theuse of photoactive, fluorescent, radioactive or enzymatic labelsrespectively are examples for photometric, fluorometric, radiologicaland enzymatic detection methods. An example of a thermodynamic detectionmethod is isothermal titration calorimetry.

In one embodiment the detection is based on an antibody specific forRUNX3. Accordingly, the absence of the RUNX3 protein is determined byusing such an antibody. Where a respective immunoglobulin or a fragmentthereof is used, it may be generated by methods well known in the art,such as phage display (cf. also Osaki et al. (2004), European Journal ofClinical Investigation 34, 605-612), or may be obtained from commercialsources. Polyclonal immunoglobulins specific for RUNX3 are for exampleavailable under the product code “ab11905” from Abcam or NovusBiologicals, under the catalogue number CBFA31-A from Acris and underthe catalogue number GTX11905 from GeneTex.

The term “antibody” as used herein, is understood to include animmunoglobulin and an immunoglobulin fragment that is capable ofspecifically binding a selected protein, e.g. RUNX3 or a protein encodedby an intestinal or a gastric marker gene, as well as a respectiveproteinaceous binding molecule with immunoglobulin-like functions.Examples of (recombinant) immunoglobulin fragments are Fab fragments, Fvfragments, single-chain Fv fragments (scFv), diabodies or domainantibodies (Holt, U et al. (2003) Trends Biotechnol. 21(11), 484-490).An example of a proteinaceous binding molecule with immunoglobulin-likefunctions is a mutein based on a polypeptide of the lipocalin family.See for example Beste et al. (1999) Proc. Natl. Acad. Sci. USA 96,1898-1903 and international patent applications WO 99/16873, WO00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO2005/019255 or WO 2005/019256. Lipocalins described in these referencessuch as the bilin binding protein, the human neutrophilgelatinase-associated lipocalin, human Apolipoprotein D, human tearlipocalin, or glycodelin, posses natural ligand-binding sites that canbe modified so that they bind to selected small protein regions known ashaptens. Other non-limiting examples of further proteinaceous bindingmolecules are the so-called glubodies (see WO 96/23879), proteins basedon the ankyrin scaffold (Hryniewicz-Jankowska, A et al., FoliaHistochem. Cytobiol. 40, 2002, 239-249) or crystalline scaffold (WO01/04144,) the proteins described in Skerra, J. Mol. Recognit. 13, 2000,167-187, and avimers. Avimers contain so called A-domains that occur asstrings of multiple domains in several cell surface receptors(Silverman, J, et al., (2005) Nature Biotechnology, 23, 1556-1561).

In one embodiment of the invention, the loss of expression of the RUNX3gene is detected by measuring the degree of methylation of the exon 1region of the RUNX3 region, wherein the term “exon” refers to a nucleicacid sequence encoding an amino acid sequence. Previously, it has beendemonstrated that RUNX3 is a major growth regulator of gastricepithelial cells that is not expressed in human gastric cancer cells dueto a hypermethylation of specific sequence regions termed “CpG islands”located within exon 1 of the RUNX3 gene (Li, Q. L. et al. (2002),supra). The methylation status of CpG islands can be assayed, forexample, by methylation-specific PCR (Herman, J. G. et al. (1996) Proc.Natl. Acad. Sci. USA 93, 9821).

The present method furthermore includes detecting the expression of oneor more intestinal marker genes. The terms “intestinal marker gene” and“gastric marker gene”, as used herein, are understood in the context ofthe gastrointestinal tract. Accordingly, an intestinal marker gene is agene that, within the gastrointestinal tract, is normally only expressedin detectable amounts—when detected by standard methods of the art—inthe gut, but for instance not in the stomach. A gastric marker gene is agene that, within the gastrointestinal tract, is normally only expressedin detectable amounts (using standard methods of the art) in thestomach, but for instance not in the gut. In some embodiments the term“intestinal marker gene” refers to any gene, which is normallyspecifically expressed in intestinal cells and tissues, and theexpression of which is indicative of an intestinal cell phenotype. Insome embodiments the term “gastric marker gene” refers to any gene,which is normally specifically expressed in gastric cells and tissues,and the expression of which is indicative of an intestinal cellphenotype. In the context of the present invention, “intestinal markergene” particularly refers to intestine-specific genes, the expression ofwhich is aberrantly induced in gastric cells having a predisposition todevelop an intestinal phenotype. Examples of intestinal marker genesinclude, but are not limited to, MUC2 (e.g. Genbank accession number ofa human mRNA: L21998), CDX2 (e.g. Genbank accession number of a humanmRNA: NM_(—)001265), PCNA (e.g. Genbank accession numbers of two humanmRNAs: NM_(—)002592 and NM_(—)182649) and the guanylyl cyclase C gene(e.g. Genbank accession number of a human mRNA: U20230).

MUC2 is a member of the family of mucins—heavily glycosylated proteinsthat are the major components of the mucous viscous gel covering thesurface of epithelial tissues (Reis, C. A. et al. (1999) Cancer Res. 59,1003-1007). MUC2 is not expressed in normal gastric mucosa, but ratherin intestinal goblet cells. However, in INTESTINAL METAPLASIA MUC2expression is observed both in globet and columnar cells.

CDX2 is a gene expressed in intestinal epithelium, regulating cellproliferation and differentiation. A ParaHox gene encoding thetranscription factor CDX2 is a mammalian homolog of Drosophila caudal,which is involved in anterior-posterior patterning. CDX2 is expressed insmall and large intestines, but not in stomach and esophagus. However,CDX2 is aberrantly expressed in the human stomach in INTESTINALMETAPLASIA, dysplasia and carcinoma (Seno, H. et al. (2002) Int. J.Oncol. 21, 769-774; Kim, H.-S. et al. (2006)Journal of Gastroenterologyand Hepatology 21, 438-442). The respective expression levels graduallyincrease from low-grade dysplasi to adenocarcinoma (Kim et al., 2006,supra). The expression of CDX2 in gastric epithelial cells of transgenicmice induces differentiation into intestinal cells (Silberg, D. G. etal. (2002) Gastroenterology 122, 689-696; Mutoh, H. et al. (2002)Biochem. Biophys. Res. Commun. 294, 470-479). Thus, CDX2 is thought tobe a major inducer of the intestinal phenotype in the gut epithelium.Without the intent of being bound by theory, it is believed that thereis a mechanism by which CDX2 is triggered to be expressed in stomach toinduce intestinal metaplasia. The inventors' findings show that CDX2 isnegatively regulated by RUNX3 and, in the absence of RUNX3, CDX2 isinduced to be expressed in gastric epithelium (Fukamachi, H et al.(2004) Biochem. Biophys. Res. Commun. 321, 58-64, incorporated herein byreference in its entirety). As a consequence, a massive expression ofintestinal markers is observed in the gastric epithelial cells of RUNX3knockout mice. As explained below, the applicants identified twodistinct levels of CDX2 expression in intestinal metaplasia. Intestinalmetaplasia with the lower expression level of CDX2 is found to beparticularly strongly associated with the development of gastric cancer.

The proliferating cell nuclear antigen (PCNA) was originallycharacterized as a DNA sliding clamp for replicative DNA polymerases andas an essential component of the eukaryotic chromosomal DNA replisome.However, subsequent studies have revealed its striking ability tointeract with numerous partners, which are involved inter alia in DNArepair, chromatin remodeling, cell cycle regulation (Maga, G. andHübscher, U. (2003) J. Cell Sci. 116, 3051-3060). Importantly, a directinteraction between PCNA and Cdk2 was detected which targetsPCNA-interacting proteins for phosphorylation (Koundrioukoff, S. et al.(2000) J. Biol. Chem. 275, 22882-22887).

Guanylyl cyclase C is a member of the family of membrane bound guanylylcyclases. serves as the receptor for the homologous diarrheagenicheat-stable enterotoxin produced by bacteria as well as for themammalian hormones guanylin and uroguanylin. In the intestinal tract,the expression of guanylyl cyclase C, which is regulated by CDX2, isrestricted to intestinal epithelial cells. Guanylyl cyclase C hasmeanwhile been found to be expressed on adenocarcinomas arising withinintestinal metaplasia in the stomach (Birbe, M. et al. (2005) HumanPathology 36, 170-179).

In other embodiments of the invention, the loss of expression of theRUNX3 gene or the expression of one or more intestinal marker genes isdetermined by detecting the absence or presence of the respectivecorresponding gene products, wherein the gene product is an mRNA or aprotein. For example, an mRNA can be detected by hybridization with alabeled nucleic acid probe (DNA or RNA) and subsequent analysis usingeither Northern blotting or performing an RNAse protection assay.Alternatively, the respective cDNA can be prepared from cellular RNA(either total RNA or mRNA) by reverse transcription and analyzed for theabsence or presence of a particular nucleic acid species by PCRamplification (i.e. RT-PCR). Proteins can be conveniently detected byusing specific antibodies, which may be monoclonal or polyclonalantibodies. For visualization, the antibody may be labeled or asecondary antibody conjugated to an appropriate label may be used, whichbinds to the specific primary antibody. Detection may be done, forexample, in a Western blot-analysis or an ELISA. Suitable labels forperforming the methods of the invention include enzyme labels,radioactive labels, fluorescent labels, chromogenic labels, luminescentlabels, digoxigenin, biotin, small organic molecules, metals, metalcomplexes, and colloidal gold.

In one embodiment, the present method of the invention further includesthe detection of one or more gastric marker genes. Two illustrativeexamples of gastric marker genes, the expression of which may bedetected, are MUC6 (e.g. Genbank accession numbers of two human mRNAs:AY458-429 and AY312160) and MUC5AC (e.g. Genbank accession numbers ofsix human mRNAs: AF043909, L46721, AJ001403, AF015521, L42292 andAJ298319).

These two proteins are also a member of the family of mucins (supra) andmainly expressed in gastric mucosa. MUC5A is particularly highlyexpressed in foveolar epithelium and mucous neck cells of the antrum.MUC6 is in particular expressed in the glands of the antrum. Theadherent gastric mucous layer has been found to include alternatinglayers of MUC5AC and MUC6. Muc6 is in particular expressed in mucousneck cells and the glands of the antrum. MUC5AC is in particularexpressed in the surface foveolar or pit cells of the stomach.Interestingly, the expression both mucins has not been found to beindicative of cancer previously. While the expression of MUC6 has beenfound not to be associated with histomorphological type or withclinicopathological features of human gastric carcinomas, even asignificant reduction of MUC5AC expression has been observed in mucinousand undifferentiated carcinomas (Baldus, S. E. et al. (2002) Ann SurgOncol. 9, 9, 887-893).

Generally, there can be identified one type of intestinal metaplasia,which is particularly strongly correlated with gastric cancer. Theinventors have found that this type of intestinal metaplasia ischaracterized by relatively low levels of CDX2 expression, when comparedto other types of intestinal metaplasia, and a detectable expression ofboth gastric mucins (e.g., MUC5AC, MUC6) and intestinal mucin (e.g.MUC2). This type, which corresponds to a type of intestinal metaplasiathat is in the art described as “incomplete intestinal metaplasia”, isin a precancerous state. While the complete type is characterized by thepresence of absorptive cells, paneth cells and goblet cells secretingsialomucins and is similar to the small intestinal phenotype, incompleteintestinal metaplasia is characterized by the presence of columnar andgoblet cells secreting sialomucins and/or sulphomucins, similar to thecolonic phenotype. The two types of intestinal metaplasia can bedistinguished by standard histology, histochemistry or mucinimmunohistochemistry methods. The inventors furthermore found thatalthough the incomplete intestinal metaplasia morphologically resemblescolon, its CDX2 expression is significantly lower than that in normalcolon.

Different types of cells in each gastric gland are believed to derivefrom a single progenitor cell. The uniformity of decreased CDX2expression in every cell in the incomplete intestinal metaplasia glands(see also Tab. 1 and Tab. 2 below) indicates that the difference iscaused by the different differentiation of the gastric progenitor cells.The inverse relationship between the expression of CDX2 and theexpression of gastric mucins (Muc5AC and Muc6) was also found in bothintestinal type and diffuse type of gastric cancer. Without intending tobe bound by theory, this finding indicates that this relationship ismaintained from intestinal metaplasia to gastric cancer. Thus, thedecrease of CDX2 in intestinal metaplasia is a useful indicator of anincreased risk of gastric cancer.

Where the expression of RUNX3 in this type of intestinal metaplasia(“incomplete”) is reduced (see FIG. 8C for an example), cells becomedysplastic and eventually develop into cancer cells. Accordingly, thereis in particular a strong correlation between lower levels of both CDX2and RUNX3 expression in intestinal metaplasia and a precancerous stateof the gastric epithelial cells. It is noted that this type ofintestinal metaplasia is always detected using the present method of theinvention, regardless of whether the expression of gastric marker genesis detected or not. Where it is desired to further characterize thepredisposition of cells to develop an intestinal phenotype (for examplein order to estimate the likeliness of a cancerous state developing),the expression of both intestinal and gastric marker genes may bedetected. As an example, the loss of expression of the RUNX3 gene aswell as the expression of the intestinal marker gene MUC2, the gastricmarker gene MUC5AC and the expression of the intestinal marker gene CDX2may be detected.

Typically, an intestinal metaplasia comprises at least a part orsubfraction that can be identified as the type described above(“incomplete intestinal metaplasia”). This type of intestinalmetaplasia, including a respective part or subfraction, may be of anyratio to an entire intestinal metaplasia. In some embodiments, such apart or subfraction is identical with the entire intestinal metaplasia.In other embodiments a respective part or subfraction is a major part ofthe intestinal metaplasia, for instance between 50 and 100% thereof. Inother embodiments it is only a minor part of the intestinal metaplasia,such as below 50% thereof.

The inventor's findings have in the meantime found support by data ofBabu, S. D. et al (2006) Molecular Cancer 5, 10,doi:10.1186/1476-4598-5-10, who report decreased expression levels ofMUC5AC and MUC6 in INTESTINAL METAPLASIA and dysplasia, as well as a denovo expression of MUC2.

In some embodiments of the present method the invention (as definedabove), the result of the measurement obtained in detecting the loss ofexpression of the RUNX3 gene is compared with a corresponding resultobtained in a control cell. The present applicants have shown thatexpression of RUNX3 is downregulated in intestinal metaplasis andgastric carcinoma (Osaki, M. (2004), supra).

In some embodiments the result of the measurement in detecting theexpression of one or more intestinal marker genes is compared to acorresponding result obtained in a control cell. Likewise, the resultobtained by measurements of detecting the expression of one or moregastric marker genes may be compared to results of measurements in acontrol cell. In some embodiments both detecting the loss of expressionof the RUNX3 gene and detecting the expression of one or more intestinalmarker genes are compared to the results of respective measurements of acontrol cell. In one embodiment all above measurements, i.e. detectingthe loss of expression of the RUNX3 gene, detecting the expression ofone or more intestinal marker genes, and detecting the expression of oneor more gastric marker genes, are compared to corresponding resultsobtained using a control cell.

The term “control cell” as used herein refers to a wild-type cell havingno predisposition to develop an intestinal phenotype.

In a second aspect, the invention provides a method for identifying acompound that inhibits, or is capable of inhibiting, the development ofan intestinal phenotype in one or more cells that have a predispositionto develop an intestinal phenotype. The cells are characterized by theloss of expression of the RUNX3 gene and the expression of one or moreintestinal marker genes as defined above. The method includes contactingthe one or more cells with a solution supposed to contain at least onecompound to be identified. The method further includes incubating thecells for a predetermined period of time. The method also includesmeasuring the expression of the RUNX3 gene as well as the expression ofthe respective one or more intestinal marker genes (cf. above).

In some embodiments the inventive method further includes comparing theresult of the measurements obtained in detecting the loss of expressionof the RUNX3 gene and the expression of one or more intestinal markergenes with corresponding results obtained in a control measurement. Sucha control measurement is carried out without the addition of thesolution supposed to contain the at least one compound to be identified.The method also includes identifying the compound that inhibits, or iscapable of inhibiting, the development of the intestinal phenotype.

The term “compound” as used herein includes any compound inhibiting thedevelopment of an intestinal phenotype regardless whether the compoundaffects the expression of the RUNX3 gene and/or the expression of one ormore intestinal marker genes. It is also understood that such compoundsmay affect any step of gene expression including transcription, RNAprocessing or translation or may interfere with the cellular function(s)of the respective proteins. Examples for such compounds aretranscriptional enhancers or repressors, RNA-binding proteins, RNAses,factors regulating the methylation status of the RUNX3 gene sequence,anti-sense nucleic acids, small organic molecules or dominant-negativemutant proteins. Examples of compounds that may be obtained using themethod of the present invention are disclosed, for example, ininternational patent application WO 2005/115388.

Any number of steps of the present method of the invention, includingthe entire method, may be performed in an automated way—also repeatedly,using for instance commercially available robots. As an illustrativeexample, the method may be an in-vitro screening method, for examplecarried out in multiple-well microplates (e.g. conventional 48-, 96-,384- or 1536 well plates) using automated work stations. The method mayalso be carried out using a kit of parts, for instance designed forperforming the present method.

Exemplary Embodiments of the Invention

Exemplary embodiments of a method for identifying cells having apredisposition to develop an intestinal phenotype are shown in theappended figures and explained in the following.

FIG. 1 depicts chief cells and surface epithelial cells, which areprevented from differentiating in the stomach of Runx3−/− mice. (a)shows a diagram of a gastric gland. (b-e) depict the immunohistochemicaldetection of surface epithelial cells with an anti-MUC5ACimmunoglobulin, wherein (d) and (e) represent enlargements of the boxedregions in (b) and (c), respectively. (f-i) show the immunohistochemicaldetection of chief cells with an anti-pepsinogen immunoglobulin, wherein(h) and (i) represent enlargements of the boxed regions in (f) and (g),respectively. (j-o) Immunohistochemical detection of chief cells (j, k),mucous neck cells (l, m) and parietal cells (m, o) with anti-pepsinogen,anti-MUC6 and anti-H⁺/K⁺ ATPase antibodies, respectively. (j-o)represent serial sections. (p) depicts the average numbers of chiefcells, mucous neck cells and parietal cells in the fundic gland of 6months-old WT and RUNX3−/− mice. (b-o) shows fundic glands of 6months-old mice. Counter staining was done with hematoxylin (b-o). Scalebars are equal to 50 μm (d, e, h-o) and 100 μm (b, c, f, g),respectively.

FIG. 2 depicts the cellular proliferation in the Lo (cf. FIG. 1) andinhibition of apoptosis in the Up (cf. FIG. 1) of Runx3−/− gastricepithelium. (a, b) depict the detection of replicating cells by5-bromo-2-deoxyuridine (BrdU) incorporation. (c, d) depict the detectionof proliferating cells by immunostaining with an anti-PCNAimmunoglobulin. (e, f) depict the detection of apoptotic cells using aDNA fragmentation detection assay. Arrowheads in (e) show apoptoticcells. (a-f) show fundic glands of 6 months-old mice. Counter stainingwas done with hematoxylin (e, f). Scale bars are equal to 50 μm (e, f)and 100 μm (a-d), respectively.

FIG. 3 depicts intestinalization of RUNX3−/− gastric epithelial cells.(a, b) depict immunohistochemical detection of CDX2 in the fundic glandsof 6 months-old mice. (c, d) show immunostaining with an anti-MUC6immunoglobulin, followed by Alcian Blue staining of 3 months-old gastricepithelia at the fundic area. MUC6-positive mucous neck cells arestained brown and thus appear dark. (e, f) depicts Alcian Blue stainingof 6 months-old gastric epithelia at the fundic area. (g, h) representenlargements of the respective boxed regions in (f). (i, j) showImmunohistochemical detection of ITF in the fundic glands of 6 month-oldmice. (k, l) depict immunohistochemical detection of MUC2 in fundicglands of 6 months-old mice. (m-o) show immunohistochemical detection ofvillin in wild-type (m) and RUNX3−/− Up (n) and Lo (o) fundic glands of6 months-old mice. (p-s) depict Immunohistochemical detection of ITF inthe pyloric glands of 6 months-old mice, wherein (r) and (s) representenlargements of the boxed regions in (p) and (q), respectively. Counterstaining was done with hematoxylin (c, d, i-s) as well as nuclear fastred (e-h). Scale bars are equal to 50 μm (a-d, g, h, p-s) and 100 μm (e,f, i-o), respectively.

FIG. 4 depicts Alcian Blue- and MUC6-positive cells with the capacity toproliferate from dysplasia in RUNX3−/− gastric epithelium. (a, e, f,)Immunostaining with an anti-MUC6 immunoglobulin, followed by Alcian Bluestaining. (b) Immunodetection of MUC6-expressing cells. (c) shows doublestaining with Alcian Blue and an anti-CDX2 immunoglobulin. (d) depictsthe detection of proliferating cells by immunostaining with an anti-PCNAimmunoglobulin. (b-d) represent serial sections, which are perpendicularto the glands in the Lo. (f) represents an enlargement of the boxedregion in (e). (g) shows hematoxylin and eosin staining performed on aserial section from (f). (c) shows strong (arrowheads) and weak (arrows)expression of CDX2 in Alcian Blue-positive cells. (g) shows dividing ordivided cells indicated by arrowheads. (a-d) are from the fundic glandsof 6 months-old mice and (e-g) from dysplasia observed in the fundicglands of 10 months-old mice. Counter staining was done with hematoxylinin (b). Scale bars are equal to 50 μm (b, f) and 100 μm (a, e),respectively.

FIG. 5 illustrates a model of the role of Runx3 in gastriccarcinogenesis. Thereby, mucous neck cells are blocked fromdifferentiating into chief cells. At the same time, the lineage ofdifferentiation is deregulated and these cells are induced to expressintestinal (I) markers, although they continue to express a gastric (G)marker. That is, the mucous neck cells accumulating in the fundic glandsin the absence of RUNX3 are no longer genuine gastric or intestinalcells. The appearance of both the gastric (G) and intestinal (I)phenotypes in a single cell or in cells of a single gastric gland isoften observed for INTESTINAL METAPLASIA of the human stomach. Togetherwith their high capacity for proliferation, the developmentally blockedabnormal mucous neck cells expressing both G and I markers observed inthe RUNX3−/− stomach are likely to be pre-cancerous. Thus, RUNX3 is notonly a major regulator of gastric epithelial cell growth but also aregulator of gastric epithelial cell differentiation.

FIG. 6 depicts intestinal metaplasia. MUC2 is intestinal mucin, normallynot present in gastric epithelium. (A) shows the expression of MUC2 inthe area which represents the entire IM in this specimen: (B) depictsMUC5AC expression. Only a subfraction of intestinal metaplasia, markedby an arrow, expresses gastric mucin. (C) shows the expression of MUC6,which is primarily expressed in the deeper gland. (D) shows theexpression of CDX2. As can be seen, there are two distinct levels ofCDX2 expression. Those glands that express a lower level of CDX2 alsoexpress MUC5AC (gastric mucin) as well as MUC2 (intestinal mucin).

FIG. 7 depicts gastric dysplasia. The lower third of each photorepresents dysplasia having densely clustered nuclei. (A) shows theexpression of MUC2, (B) shows the expression MUC5AC, (C) shows theexpression of MUC6, and (D) shows the expression of CDX2. No CDX2expression was detected. Some of the dysplastic cells express bothgastric (MUC5AC, MUC6) and intestinal mucin (MUC2).

FIG. 8 depicts the expression of RUNX3 in dysplasia. The figurecorresponds to FIG. 7 with photo (C) of FIG. 7 (showing MUC6 expression)being replaced by a photo (C) showing RUNX3 expression. Expression ofRUNX3 is lower than that in most of the intestinal metaplasia and alarge fraction of RUNX3 is in the cytoplasm in dystplasia.

EXAMPLES Example 1 Block of Differentiation of Mucous Neck Cells intoChief Cells in RUNX3−/− Fundic Glands

Histological analyses were performed according to established standardprocedures well known in the art.

Tissues were fixed with 4% paraformaldehyde in PBS, embedded inparaffin, and cut into 5 μm sections. For staining, the re-hydratedsections were placed in 1% Alcian Blue in 3% acetic acid (pH 2.5) for 30min at 23° C. For immunohistochemical detections the followingantibodies were used on re-hydrated sections: anti-MUC5AC (K-20; SantaCruz, USA), anti-pepsinogen (Fukamachi, H. et al. (2001)Biochem.Biophys. Res. Commun. 280, 1069-1076), anti-MUC6 (HIK1083; Kanto Kagaku,Japan), anti-H⁺/K⁺ ATPase (1H9; MBL, USA), anti-ITF (Mashimo, H. et al.(1996) Science 274, 262-265), anti-MUC2 (Karisson, N. G. et al. (1996)Glycoconjugate J. 13, 823-831), anti-villin (MAB1671; Chemicon, USA),anti-CDX2 (BioGenex, USA), and anti-PCNA (MAB424R; Chemicon, USA).Signals were detected by consecutive incubation with biotin-conjugatedsecondary antibodies and streptavidin-FITC (Roche MolecularBiochemicals, USA).

Staining for apoptotic cells was performed by using the Klenow-FragELDNA fragmentation detection kit (Oncogene Research, USA) according tothe recommendations of the manufacturer. Proliferating cells werelabeled with 5-bromo-2-deoxyuridine (BrdU) using the BrdU Labeling andDetection Kit II (Roche Molecular Biochemicals, USA). BrdU (30 mg/kgbody weight) was injected i.v. into mice. Four hours later the excisedstomachs were fixed with 4% paraformaldehyde in PBS, embedded inparaffin, and cut into 5 μm sections.

The generation of RUNX3−/− mice has been described previously (Li, Q. L.et al. (2002), supra). RUNX3−/− mice of the C57BL/6 background die soonafter birth due to starvation. RUNX3−/− mice of the BALB/c background,on the other hand, survive for at least 10 months. Therefore, thegastric phenotype of mutant mice of the BALB/c background wascharacterized by monitoring changes in the gastric mucosa. In the fundicarea of wild type mice, a stem cell zone exists at the neck of glands.Thus, gastric mucosa could be separated into an upper (referred to as“Up”, cf. also FIG. 1 (a)) and a lower compartment (referred to as “Lo”;cf. also FIG. 1 (a)) by the zone. Cells migrating towards the lumenundergo terminal differentiation to become surface epithelial cells,which are renewed every 3 to 4 days in both mice and humans (Karam, S.M. and Leblond, C. P. (1993) Anat. Rec. 236, 280-296). Cells migratingtowards the gland base become mucous neck cells, which express MUC6 andlow levels of pepsinogen. These cells eventually differentiate intochief cells at the bottom of glands, Which do not express MUC6 but doproduce large amounts of pepsinogen (FIG. 1 (j, l)). The life span ofchief cells is around 6 months. In the stomach of wild type adult mice,RUNX3 was maximally expressed in chief cells and, to a lesser degree, insurface epithelial cells, but at reduced levels in parietal cells andmucous neck cells (Li, O. L. et al. (2002), supra).

In the gastric mucosa of 6 months-old wild type mice, pepsinogen wasfully expressed in chief cells, whereas it was detected at significantlyreduced levels in the corresponding area in RUNX3−/− mice (compare FIG.1 (g, i) with (f, h)). Instead, RUNX3−/− cells in virtually throughoutthe Lo, including the bottom area of the gland, were found to expressMUC6 (FIG. 1 (m)). These results suggest that the differentiation ofmucous neck cells into chief cells was blocked in the RUNX3−/− fundicglands. A determination of the cell number in each gland confirmed thedecrease in chief cells and increase in mucous neck cells in mutant mice(FIG. 1 (p)). The cytoplasm of MUC6-expressing cells in the Lo ofRUNX3−/− mucosa was filled with large amounts of mucin, and the cellswere enlarged (FIG. 1 (m)).

Parietal cells secreting hydrochloric acid are also present in the Lo.In RUNX3−/− mice, they appeared smaller and morphologically somewhatdistorted compared with those in wild type mice (compare FIG. 1 (o) with(n)). Since mucous neck cells are often enlarged in mutant mice due to alarge amount of mucin in the cytoplasm, parietal cells appeared to bepressed between the mucous neck cells (compare FIG. 1 (o) with (n)).However, the number and the expression of proton pumps characteristic ofparietal cells did not seem to be affected by the absence of the RUNX3(FIG. 1 (p)). A reduction in the acidity of stomach epithelium caninduce INTESTINAL METAPLASIA (Stemmermann, N. G. (1994), supra).Therefore, it was important to determine the acidity of the gastricmucosa of the mutant mice to confirm whether the parietal cells werefunctionally intact. Stomachs of the mutant mice varied in acidity frompH 2 to 5, although about half of them were lower than pH 3.0, whilewild type stomachs were less variable, fluctuating between pH 2.0 and pH3.0. Nevertheless, it was demonstrated that an intestinal phenotype (seebelow) was induced in RUNX3−/− gastric mucosa having an acidity below pH3.0, ruling out the possibility that the induction of intestinalphenotype is due to a reduction in acidity rather than to theinactivation of RUNX3.

Example 2 Analysis of the Proliferation Capacity of Abnormally ExpandedMucous Neck Cells

Since mucous neck cells can proliferate, it was examined whether theabnormally expanded mucous neck cells throughout the Lo of mutant micehave growth potential. BrdU was found to be actively incorporated in ahigh proportion of MUC6-expressing cells, even in cells deep within theLo of RUNX3−/− mouse stomach (compare FIG. 2 (b) with (a)).Immunodetection of proliferating cell nuclear antigen (PCNA) also showedthe high capacity for proliferation of RUNX3−/− gastric epithelial cellsin the Lo (compare FIG. 2 (d) with (c)). In contrast, the majority ofcells in the Up did not incorporate BrdU or display PCNA reactivity,suggesting that Up cells were not experiencing accelerated growth.

Muc5AC is expressed in the foveolar epithelium of the stomach. The Upcells of RUNX3−/− mice showed a greatly reduced expression of Muc5ACcompared with wild type cells (compare FIG. 1 (c, e) with (b, d)). Theterminal differentiation of surface epithelial cells also appeared to beblocked in the absence of the RUNX3.

It is notable that the Up as well as the Lo mucosa of mutant miceexhibited hyperplasia (compare, for example, FIG. 2 (b, d) with (a, c)).Since in RUNX3−/− gastric epithelial cells of mice of the C57BU6background a lack of apoptosis was observed (Li, Q. L. et al. (2002),supra), it was examined whether apoptosis is reduced in BALB/c gastricepithelial cells that lack Runx3 activity. Indeed, a significantreduction in the frequency of apoptotic cells in the Up of mutant micewas observed, as compared with that in wild type mice (compare FIG. 2(f) with (e)). Since this finding could provide a possible basis for thehyperplasia observed in the Up of mutant mice, it will be necessary todetermine whether the cells in the Up can develop into cancer cells.Immunohistochemical observations demonstrated that the differentiationof endocrine cells in RUNX3−/− gastric epithelium, includinggastrin-positive cells (G cells) of the pyloric area, was not affected(data not shown).

Example 3 Expression of Intestinal Marker Genes in Gastric EpithelialCells

In RUNX3−/− mice, CDX2 was expressed throughout the gastric mucosa, bothin the Up and Lo except for parietal cells (compare FIG. 3 (b) with(a)), although the level of expression varied considerably from cell tocell (FIGS. 3 (b), 4 (c)). In addition to CDX2, several otherintestine-specific markers were examined. Intestinal trefoil factor(ITF), secreted throughout the small and large intestines, is consideredto have an important role in the maintenance and repair of theintestinal mucosa (Mashimo, H. et al. (1996), supra) and is a marker ofpoor prognosis in gastric carcinoma (Yamachika, T. et al. (2002) Clin.Cancer Res. 8, 1092-1099). MUC2 is specifically expressed in the gobletcells of the small and large intestines (Karisson, N. G. et al. (1996),supra). Alcian blue reacts with acidic mucins (i.e. sulphomucin andsialomucin; Reis, C. A. et al. (1999), supra) and is often used todetect intestinal markers. Villin is a structural protein of intestinalabsorptive cells (Dudouet, B. et al. (1987) J. Cell Biol. 105, 359-369).

A significant expression of these intestinal markers was observed in thegastric mucosa of one month-old RUNX3−/− mice. However, Alcian Bluestained the gastric mucosa of mutant mice that were 3 months old andolder (FIG. 3 (d, f)), whereas it did not stain the gastric mucosa ofwild type mice 3 months (FIG. 3 (c)) or 6 months (FIG. 3 (e)) afterbirth. The earliest stage when Alcian Blue-positive cells were observedin RUNX3−/− gastric mucosa was around 10 weeks, and the maximal levelsof RUNX3 expression in the gastric epithelial cells of wild type micewas observed after 8-10 weeks (data not shown). Up and Lo cells in 6months-old mice were stained by Alcian Blue (FIG. 3 (f-h)). MUC2 wasalso detected in both Up and Lo cells of the mutant but not wild typemice (compare FIG. 3 (l) with (k)). The intestinal phenotype was firstnoted when the glandular structure was fully formed in the mousestomach. The fact that RUNX3 is expressed most strongly in mature chiefcells and surface epithelial cells suggests that it begins to exert itscritical function around the time the gland structure is fully formed.Therefore, the RUNX3−/− phenotype may become evident around the time ofgland maturation. Like CDX2, ITF was detected throughout 6 months-oldRunx3−/− gastric mucosa in both the Up and Lo and within the same gland(compare FIG. 3 (j) with (4). Villin is expressed strongly in the Up andweakly in the Lo of Runx3−/− gastric epithelium (compare FIG. 3 (m, o)with (m)). The expression of CD10 (Groisman, G. M. et al. (2002) Am. J.Surg. Pathol. 26, 902-907) and alkaline phosphatase in RUNX3−/− gastricmucosa was undetectable (data not shown). Since the latter two markersare specific to small intestinal absorptive cells, the majority of thecells in the RUNX3−/− gastric mucosa that express these intestinalphenotypes are considered to be similar to colon epithelial cells. Thestructure of the pyloric glands, where human gastric cancer mostfrequently develops, is simpler than that of the fundic glands, in thatonly surface epithelial cells, proliferative cells, and mucoid cellsexpressing pepsinogen are present. Intestinalization of the mucosa wasalso observed in the RUNX3−/− pyloric region, as revealed by theexpression of ITF (compare FIG. 3 (q, s) with (p, r)).

It is noteworthy that each gland of the mucosa is composed of cellsderived from a single progenitor (Tatematsu, M. et al. (2003) CancerSci. 94, 135-141). The monoclonal origin of each gland thus makes iteasier to interpret the complex phenotypes observed in knockout mice. Inthe RUNX3−/− gastric mucosa, almost all glands displayed a gastric (G)phenotype alone, an intestinal (I) phenotype alone or the mixture ofthese two (GI), which were manifested as Alcian Blue- and MUC6-positivecells (FIG. 4 (a-c)), and these cells were proliferative (FIG. 4 (d)).The appearance of the G, mixed GI, and I phenotypes appears to bestochastic in RUNX3−/− epithelial cells rather than chronological(Tatematsu, M. et al. (2003), supra). Since cells in a single glandpresumably have identical genotypes, variation in the penetrance of theG and I phenotypes may be governed by epigenetic mechanisms (see below).

Dysplastic lesions were often found in RUNX3−/− mice at 10 months ofage: the gastric epithelial cells were irregularly arranged, andcontained hyperchromatic nuclei that were disproportionately large. Somecells penetrated through the muscularis mucosae towards the submucosa(FIG. 4 (e)). These cells expressed MUC6, a G marker, and were positivefor Alcian Blue, an I marker (FIG. 4 (e, f)). They were highlyproliferative (FIG. 4 (g)). These results indicate that cells exhibitingthe mixed GI phenotype in the RUNX3−/− gastric epithelium at around 6months of age are pre-cancerous in nature, and these cells undergodysplastic changes presumably by acquiring further genetic or epigeneticchanges.

From the results described above, the observations can be summarized inthe model shown in FIG. 5. Mucous neck cells are blocked fromdifferentiating into chief cells. At the same time, the lineage ofdifferentiation is deregulated and these cells are induced to express Imarkers, although they continue to express a G marker. That is, themucous neck cells that accumulate in the glands in the absence of Runx3are no longer genuine gastric or intestinal cells. The appearance ofboth the G and I phenotypes in a single cell or in cells of a singlegastric gland is often observed for INTESTINAL METAPLASIA of the humanstomach. Together with their high capacity for proliferation, thedevelopmentally blocked abnormal mucous neck cells expressing both G andI markers observed in the RUNX3−/− stomach are typically pre-cancerous.Indeed, the dysplasia observed in 6 to 10 month-old mutant miceexhibited the mixed GI phenotype. Cells destined to become surfaceepithelial cells are also unable to terminally differentiate and areresistant to apoptosis. Thus, it is feasible that developmentallyblocked surface epithelial cells become cancer cells. Previously, it wasdemonstrated that RUNX3 is a major growth regulator of gastricepithelial cells (Li, Q. L. et al. (2002), supra). Here, evidence isprovided that the gene is also a regulator of gastric epithelial celldifferentiation.

A morphological hallmark of human INTESTINAL METAPLASIA is the presenceof readily recognizable goblet cells. In the “intestinalization” in theRUNX3−/− gastric mucosa shown here, very few typical goblet cells wereobserved, despite the fact that widespread expression of intestinalmarkers including MUC2 was detected in these cells. There is a distinctdifference between the inactivation of RUNX3 in the germ line ofRUNX3−/− gastric mucosa and RUNX3 inactivation in human INTESTINALMETAPLASIA where it is a somatic event. In order for cells to acquiregoblet cell morphology, RUNX3 might be required at certain stages indevelopment. Goblet cell formation fails to occur in the absence of theMUC2 gene (Velcich, A. et al. (2002) Science 295, 1726-1729), the gene,which encodes the zinc-finger transcription factor Klf4 (Katz, J. P. etal. (2002) Development 129, 2619-2628), or the Math1, gene encoding abasic helix-loop-helix (bHLH) transcription factor (Yang, Q. et al.(2001) Science 294, 2155-2158). Math1, is also required for endocrinecell formation in the intestine but such cells were not affected in thestomach of RUNX3−/− mice (data not shown). Since MUC2 and Klf4 seem tobe downstream targets of CDX2 (Dang, D. T. et al. (2001) Oncogene 20,4884-4890; Mesquita, P. et al. (2003) J. Biol. Chem. 278, 51549-51556)and as both CDX2 and MUC2 are expressed in the stomach of RUNX3−/− mice,failure to form goblet cells does not seem to be due to a lack of Klf4expression. RUNX3 may thus be required for other pathways in goblet cellformation.

In any event, it will be important to determine whether the appearanceof goblet cells in gastric mucosa per se is causally related to gastriccarcinogenesis or whether a more general induction of intestine-specificgene expression is required to induce pre-cancerous conditions.Nevertheless, the dramatic induction of intestine-specific genes in bothcases warrants a direct comparison of the phenotypes of RUNX3−/− gastricmucosa and INTESTINAL METAPLASIA. The inactivation of RUNX3 is thussuggested to induce INTESTINAL METAPLASIA. Considering the fact thatRUNX3 is a candidate for a gastric cancer tumor suppressor gene,INTESTINAL METAPLASIA must be a pre-cancerous state, wherein theinactivation of RUNX3 appears to trigger the carcinogenesis process.

The mechanism of gastric carcinogenesis triggered by the inactivation ofRUNX3 suggested above is strikingly similar to that observed for RUNX1inactivation. RUNX1 is required for the granulocytic differentiation ofmyeloid progenitor cells, and its inactivation is leukemogenic(Sakakura, C. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11723-11727).This observation is in line with the more general notion that the normalphysiological functions of most oncogenes and tumor suppressor genes areto regulate differentiation and that cancer is a consequence ofderegulated development and differentiation (Bishop, J. M. (1983) Cell32, 1018-1020).

However, the link between INTESTINAL METAPLASIA and cancer may be basedon complex interactions. In may cases of INTESTINAL METAPLASIAexpression of RUNX3 was observed. Toward clarifying the relationshipbetween RUNX3 expression and INTESTINAL METAPLASIA in human gastricepithelium, the expression of gastric mucins (MUC5AC, MUC6) andintestinal mucin (MUC2) we examined in relation to CDX2 expression inINTESTINAL METAPLASIA in human specimens (cf. FIG. 6). Interestingly,two distinct levels of CDX2 expression were found. Low levels of CDX2were found to correlate with previously classified incomplete INTESTINALMETAPLASIA, in which both gastric and intestinal mucins are expressed(cf. FIG. 7 and FIG. 8). This incomplete INTESTINAL METAPLASIA isconsidered to be particularly strongly associated with gastric cancer.

The observation that CDX2 is up-regulated in the gastric epithelialcells of RUNX3−/− mice suggests that RUNX3 negatively regulates CDX2,either directly or indirectly, in wild type gastric epithelial cells. Inthis regard Kim et al (2006, supra) have confirmed that there is no CDX2expression in normal mucosa, but is detectable in both INTESTINALMETAPLASIA and carcinoma. It is noted that RUNX3 is expressed inwild-type intestinal epithelial cells but at levels lower than ingastric epithelial cells (data not shown). Therefore, the level of RUNX3expression in intestinal cells may be too low to suppress CDX2.Alternatively, the sets of transcription factors expressed in intestinaland gastric epithelial cells that induce tissue-specific phenotypes maydiffer, such that the expression of CDX2 is independent of theexpression of Runx3 in intestinal epithelial cells.

While it is not known whether the aberrant expression of CDX2 in stomachcells induces a pre-cancerous state, it is known that inactivation ofthe CDX2 gene induces intestinal tumor and colonic hamartoma (Beck, F.et al. (1999) Proc. Natl. Acad. Sci. USA 96, 7318-7323; Tamai, Y. et al.(1999) Cancer Res. 59, 2965-2970). CDX2 seems to function as a tumorsuppressor in the intestine and colon, in contrast to its possiblefunction in the stomach. In this regard, it is notable that cells inINTESTINAL METAPLASIA, which are likely to be pre-cancerous, expressCDX2, whereas gastric cancer cells often do not. It appears that theexpression of CDX2 tends to be turned off in cancer cells. It is alsoworth noting that CDX2 directs endodermal differentiation towards acaudal phenotype and that haploinsufficiency in the developing distalintestine leads to homeotic transformation to a more rostral endodermphenotype (Beck, F. et al. (1999), supra). We have shown thatinactivation of RUNX3 induces transdifferentiation in the oppositedirection, from a gastric to an intestinal character. Furtherelucidation of the exact relationship between RUNX3 and CDX2 would shedlight on the development of gastrointestinal tract tumors and gastriccarcinogenesis.

A possible role for RUNX3 in lineage-specific gene expression has beenfound for T cell development (Taniuchi, I. et al. (2002) Cell 111,621-633; Woolf, E. et al. (2003) Proc. Natl. Acad. Sci. USA 100,7731-7736). Immature thymocytes lacking CD4 and CD8 co-receptorsdifferentiate into doubly positive (CD4⁺CD8⁺) cells, which are selectedto become either CD4⁺CD8⁻ helper cells or CD4−CD8⁺ cytotoxic cells. Atranscriptional silencer regulates the expression of CD4 in bothimmature and CD4−CD8+ thymocytes. It has been shown that RUNX1 isrequired for active repression in doubly negative thymocytes, whereasRUNX3 is required for establishing epigenetic silencing in cytotoxiclineage thymocytes. Since RUNX3 is specifically required forlineage-specific CD4 silencing, it may be a key molecule involved inregulating lineage specificity. In the absence of RUNX3, CD4−CD8⁺ cellsdo not arise, but CD4⁺CD8⁺ cells having a cytotoxic cell markeraccumulate. In other words, these cells express a mixture of markersderived from two lineages, strikingly similar to what is observed forgastric epithelial cells in RUNX3−/− mice. If RUNX3 is required for theformation of a chromatin structure conducive to the expression of chiefcell-specific or surface epithelial cell-specific genes, this mayexplain the failure of these cells to differentiate in RUNX3−/− mice. Itmay thus be interesting to investigate the possible roles of RUNX familygenes in the assembly and remodeling of chromatin with respect tolineage-specific cell differentiation in order to understand themechanisms by which these genes induce carcinogenic processes.

Example 4 Analysis of the Expression of Intestinal and Gastric MarkerGenes in Tissue Samples from Patients with Intestinal Metaplasia andGastric Dysplasia

surgically resected gastric adenocarcinoma samples and the correspondingnon-cancerous tissues were obtained from the Department of Pathology andSurgery, National University of Singapore under a protocol approved byInstitutional Review Board. There were 47 males and 23 females, aged31-86 years (mean±SEM, 62.6±1.6). 38 intestinal metaplasia foci werefound in the tissues of 26 cases, including 18 intestinal metaplasiafoci just beside the tumor in 16 cases and 20 intestinal metaplasia focion the non-cancerous tissues in 15 cases.

According to the Lauren-classification of gastric adenocarcinoma(Lauren, P., et al., (1965) supra), there were 45 intestinal type ofgastric adenocarcinoma and 19 diffuse type of gastric adenocarcinoma.The other 6 cases were mucinous adenocarcinoma, displaying anintestinal-type pattern of infiltration. It was listed as an individualgroup because of its unique expression pattern of mucins. The 45intestinal type of gastric adenocarcinoma were further classified intowell differentiated (14 cases), moderately differentiated (11 cases) andpoorly differentiated (20 cases).

In addition, 7 gastric dysplasia samples, 5 colon adenocarcinoma samplesand the corresponding non-cancerous tissues were obtained fromDepartment of Pathology and Surgery, National University of Singapore.

Histochemistry

The samples were fixed with 10% neutral-buffered formalin and embeddedin paraffin. Paraffin-embedded samples were serially sectioned at 4 μmand mounted on slides. intestinal metaplasia was classified intocomplete type and incomplete type, using Alcian Blue (pH 2.5)/PeriodicAcid-Schiff (AB/PAS) staining (Dako AB/PAS stain system, Dakocytomation,Carpinteria, Calif.). After deparaffinization and rehydration, thesections were incubated with Alcian Blue pH 2.5 for 30 minutes, followedby 0.5% Periodic Acid for 10 minutes and Schiff solution for 10 minutes.Then the sections were counterstained with modified Mayer's hematoxylinfor 5 minutes, dehydrated with graded ethanols and mounted withcoverslip. In complete intestinal metaplasia, only goblet cells werestained blue while the intermediate absorptive non-secretory cells werenot stained. In incomplete intestinal metaplasia, the goblet cells werestained blue and the intermediate columnar cells were also stained withblue or pink.

Immunohistochemistry

After routine deparaffinization and rehydration of the slides, theantigen retrieval was done by the incubation in modified citrate buffer(target retrieval solution, Dakocytomation, Carpinteria, Calif.) at 96°C. for 40 minutes (for Muc2, Muc5AC, Muc6 and Ki67 antibodies) or at121° C. for 20 minutes (for CDX2 antibody). During the optimization ofthe CDX2 staining protocol, it was observed that higher concentrationsof CDX2 antibody, overnight incubation of primary antibody and antigenretrieval with EDTA buffer at 121° C. for 20 minutes could significantlyenhance the CDX2 staining intensity in incomplete intestinal metaplasiaand thus diminish the difference between incomplete intestinalmetaplasia and complete intestinal metaplasia.

The sections were treated with 0.03% hydrogen peroxide for 5 minutes toblock the endogenous peroxidase activity, The sections were thenincubated with anti-CDX2 monoclonal antibody (1:100, Biogenex, SanRamon, Calif.), anti-Muc2 monoclonal antibody (1:100, Novocastralaboratories, Newcastle, UK), anti-Muc5AC monoclonal antibody (1:100,Novocastra laboratories, Newcastle, UK), anti-Muc6 monoclonal antibody(1:100, Novocastra laboratories, Newcastle, UK) or anti-Ki67 monoclonalantibody (1:100, Dakocytomation, Carpinteria, Calif.) at roomtemperature for 1 hour. The sections were incubated with peroxidaselabeled polymer from Envision+ System-HRP (DAB) kit (Dakocytomation,Carpinteria, Calif.) at room temperature for 1 hour. After developmentwith diaminobenzedine, the sections were counterstained withhematoxylin, dehydrated with graded ethanols and mounted with coverslip.

Assessment of Staining in Cancer

At least 10 representative fields under high power magnification (×400)were chosen and more than 1000 cancer cells or, if the total number ofcancer cells was below 1000, all available cancer cells were counted foreach section.

The Ki67 proliferation index (Ki67 is an antigen detecting proliferatingcells in the G1-, S- and G2-phase of the cell cycle) was defined as thepercentage of Ki-67 positive cancer cells. Both qualitative andsemi-quantitative approaches were used in scoring the staining of Muc2,Muc5AC, Muc6 and CDX2. Samples were classified as positive if >5% tumorcells stained positive and otherwise as negative.

Semi-quantitative scores were given as the score of percentage ofpositive cells plus the score of the staining intensity. The scoringcriteria of the percentage of positive cells were as follows: 0, 0-5%positive cancer cells; 1, 6-25% positive cancer cells; 2, 26-50%positive cancer cells; 3, 51-75% positive cancer cells; 4, 76-100%positive cancer cells. The intensity score was given as follows: 0, nostaining; 1, weak/equivocal staining; 2, mild staining; 3, moderatestaining; 4, strong staining. The minimum score was 0 and the maximumscore was 8.

Two experienced investigators independently examined the staining whileblind to the clinicopathologica data. Different scores between the twoinvestigators were observed in <15% of the cases and a consensus couldbe achieved in all the cases after discussion.

Statistical Analysis

The Fisher's exact test or the Chi-square test was used to calculate thedifference of distribution in two or three groups. T-test or one-wayANOVA with bonferroni test was used to compare the means between 2groups or among 3 groups. Difference at P<0.05 were considered to bestatistically significant.

Results The Expression of Muc2, Muc5Ac, Muc6 and CDX2 in Normal GastricMucosa and Intestinal Metaplasia

In normal gastric mucosa, Muc5Ac was expressed in the superficialfoveolar epithelium and Muc6 was expressed in the mucous neck cells ofthe body and deeper glands of the antrum. Muc2 and CDX2 are notexpressed in the normal gastric mucosa (Table 1, see below).

38 foci of intestinal metaplasia were classified into 19 foci ofcomplete type and 19 foci of incomplete type by AB/PAS staining. Muc2was expressed in the goblet cells in all intestinal metaplasia glands.Muc5AC was expressed in both goblet cells and columnar cells inincomplete intestinal metaplasia. It was also expressed in a few gobletcells but not absorptive cells in complete intestinal metaplasia. Muc6was expressed in both goblet cells and columnar cells in several deeperglands of incomplete intestinal metaplasia (data not shown). This is inagreement with previous reports which showed complete intestinalmetaplasia with de novo expression of intestinal mucin-Muc2 anddecreased expression of gastric mucins while incomplete intestinalmetaplasia with co-expression of intestinal and gastric mucins. In a fewglands within the foci of complete intestinal metaplasia, Muc5AC andMuc6 were expressed in both goblet cells and columnar cells and thisdemonstrated the ‘mosaic’ pattern of intestinal metaplasia subtypes.

CDX2 expression was significantly lower in the foci of incompleteintestinal metaplasia than in the foci of complete intestinalmetaplasia. Furthermore, within the mosaic foci of intestinalmetaplasia, the CDX2 expression in the glands which expressed Muc5AC orMuc6 was significantly lower than the glands which did not expressMuc5AC and Muc6 (data not shown). This indicates the decrease of CDX2expression is consistent in every gland of incomplete intestinalmetaplasia (cf. also FIG. 6).

The Expression of CDX2 in Normal Colon and Colon Cancer

The CDX2 protein was strongly expressed in normal colon epithelium, fromsuperficial to deeper glands. 2 out of 5 colon cancer cases testedshowed loss or significantly decreased CDX2 expression in cancer tissuecompared with normal colon epithelium (data not shown). This furthershows the tumor-suppressive role of CDX2 in colon. Considering the factthat incomplete intestinal metaplasia is associated with greater risk ofgastric cancer compared with complete intestinal metaplasia, thedecrease of CDX2 in incomplete intestinal metaplasia may be important ingastric carcinogenesis. The further decrease or loss of CDX2 in themajority of gastric dysplasia and gastric cancer further illustrates thetumor-suppressive role of CDX2 in stomach as well as in colon.

The Expression of Muc2, Muc5Ac, Muc6 and CDX2 in Gastric Cancer

The results were summarized in Table 2. The Muc2 protein was expressedin 100% mucinous type of gastric cancer (6/6, score 7.17±0.54) andsignificantly higher than that in intestinal type (51%, 23/45, score3.24±0.49) (P<0.05).

The CDX2 score of the gastric cancers with positive Muc2 expression wassignificantly higher than that of the gastric cancers with negative Muc2expression (P<0.001). However, the CDX2 score of the gastric cancerswith positive Muc5AC or Muc6 expression was significantly lower thanthat of the gastric cancers with negative Muc5AC or Muc6 expressionrespectively.

Using Muc5AC and Muc6 as the markers of gastric differentiation and Muc2as the marker of intestinal differentiation, The gastric cancer wereclassified into four catogories: G, positive for one or both gastricmucins (Muc5AC and Muc6) only; G1, positive for intestinal mucin (Muc2)and at least one of the gastric mucins (Muc5AC and Muc6); I, positivefor intestinal mucin (Muc2) only; N, none of the Muc2, Muc5AC and Muc6is positive. The 70 gastric cancer cases were classified into 20 casesof G type, 26 cases of G1 type, 14 cases of I type and 10 cases of Ntype. The CDX2 score in I type of gastric cancer was the highest amongthe four groups, which is significantly higher than that in G1 type ofgastric cancer. The latter was also significantly higher than that in Gtype of gastric cancer.

The Expression of Muc2, Muc5Ac, Muc6 and CDX2 in Gastric Dysplasia

Out of the 7 biopsies of gastric dysplasia, only 1 case ofnon-intestinal metaplasia dysplasia showed marked CDX2 expression. Inthe other 6 dysplastic intestinal metaplasia cases, 3 cases showed lostand the other 3 cases showed significantly decreased CDX2 proteinexpression. The non-intestinal metaplasiadysplasia was just next to theincomplete IM glands and 5 out of the 6 dysplastic intestinal metaplasiacases showed Muc 5AC and/or Muc6 expression in the dysplastic glands,suggesting the close relationship between dysplasia and incompleteintestinal metaplasia (FIG. 7).

The Ki67 Expression in Normal Gastric Mucosa, Intestinal Metaplasia andGastric Cancer

Ki67 positive cells were found in the neck region of normal gastricmucosa. Ki67 was also expressed in some cells in deeper glands ofintestinal metaplasia, with no significant difference between completeintestinal metaplasia and incomplete intestinal metaplasia. The Ki67index of cancer was not significantly related with theclinicopathological data and the expression of Muc2, Muc5AC, Muc6 andCDX2.

Correlation of the Expression of CDX2 and Mucins withClinicopathological Features

The age of the patients with intestinal type of gastric cancer(66.2±1.6) was significantly higher than that of the patients withdiffuse type of gastric cancer (54.6±3.7) (P<0.01). There was nosignificant association between the expression of Muc2, Muc5AC, Muc6 andCDX2 and the clinipathological data, including age and sex.

The listing or discussion of a previously published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge. All documents listed are hereby incorporated herein byreference in their entirety.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by exemplary embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

TABLE 1 The expression pattern of Muc2, Muc5Ac, Muc6 and CDX2 in normalgastric mucosa, complete IM, incomplete IM and normal colonic mucosaMuc2 Muc5AC Muc6 CDX2 Normal gastric mucosa — +in superficial +in themucous neck cells of the body — foveolar epithelium and deeper glands ofthe antrum Complete IM +in goblet cell −or +in few goblet — Stronglypositive in both goblet cells cells and columnar cells Incomplete IM +ingoblet cell +in both goblet cells +in both goblet cells and columnarWeakly positive in both goblet and columnar cells cells cells andcolumnar cells Normal colonic mucosa +in goblet cell — — Stronglypositive in every cell

TABLE 2 The expression of Muc2, Muc5AC, Muc6 and CDX2 in gastric cancerMuc2 Muc5AC Muc6 CDX2 Positive Positive Positive Positive Gastric cancertype cases (%) Score* cases (%) Score* cases (%) Score* cases (%) Score*Intestinal Total (45)# 23 (51%){circumflex over ( )} 3.24 ±0.49{circumflex over ( )} 23 (51%) 3.47 ± 0.53 18 (40%)  2.22 ± 0.42 30(67%) 3.20 ± 0.39 type Differentiation Well (14) 10 (71%) 4.57 ± 0.85  7(50%) 3.36 ± 0.96 5 (36%) 2.07 ± 0.79 11 (79%) 4.00 ± 0.69 Moderate  4(36%) 2.09 ± 0.88  7 (64%) 4.27 ± 1.05 5 (45%) 2.36 ± 0.82  6 (55%) 2.27± 0.73 (11) Poor (20)  9 (45%) 2.95 ± 0.77  9 (45%) 3.10 ± 0.81 8 (40%)2.25 ± 0.64 13 (65%) 3.15 ± 0.61 Diffuse type (19) 11 (58%) 3.95 ± 0.8214 (74%) 5.42 ± 0.79 8 (42%) 2.63 ± 0.75  8 (42%) 2.37 ± 0.68 Mucinoustype (6)  6 (100%){circumflex over ( )} 7.17 ± 0.54{circumflex over ( )} 3 (50%) 3.83 ± 1.72 0 (0%)  0 ± 0  5 (83%) 4.33 ± 0.99 Total (70) 40(57%) 3.77 ± 0.41 40 (57%) 4.03 ± 0.43 26 (37%)  2.14 ± 0.34 43 (61%)3.07 ± 0.33 #The figure in the brackets is the number of cases. *Scoreswere expressed as the mean ± the standard error of the mean {circumflexover ( )}P < 0.05 when compared with the other in the same column

1. A method for identifying one or more cells having a predisposition todevelop an intestinal phenotype, the method comprising detecting in saidone or more cells: (a) the loss of expression of the RUNX3 gene; and (b)the expression of one or more intestinal marker genes.
 2. The method ofclaim 1, further comprising: (c) comparing the result of themeasurements obtained in steps (a) and (b) with those obtained in acontrol cell.
 3. The method of claim 1, wherein the one or more cellsare epithelial cells.
 4. The method of claim 3, wherein the cells aregastric epithelial cells.
 5. The method of claim 4, wherein the cellsare gastric mucous neck cells.
 6. The method of claim 1, wherein theintestinal phenotype is characterized by a retained capacity toproliferate.
 7. The method of claim 6, wherein the intestinal phenotypeis a precursor of gastric cancer.
 8. The method of claim 6, wherein theintestinal phenotype is INTESTINAL METAPLASIA.
 9. The method of claim 1,wherein the loss of expression of the RUNX3 gene is detected bymeasuring the degree of methylation of the exon 1 region of the RUNX3gene and/or determining the absence of a RUNX3 gene product, wherein thegene product is selected from the group consisting of an mRNA and aprotein.
 10. The method of claim 9, wherein the absence of the RUNX3protein is determined by using a specific antibody.
 11. The method ofclaim 1, wherein the one or more intestinal marker genes are selectedfrom the group of genes encoding a member of the group consisting ofMUC2, and CDX2.
 12. The method of claim 1, wherein (b) further comprisesdetecting the expression of one or more gastric marker genes.
 13. Themethod of claim 12, wherein the one or more gastric marker genes areselected from the group of genes encoding a member of the groupconsisting of MUC6 and MUC5AC.
 14. The method of claim 12, wherein theloss of expression of the RUNX3 gene, the expression of the intestinalmarker genes MUC2 and CDX2, and the expression of the gastric markergene MUC5AC is detected.
 15. The method of claim 1, wherein theexpression of the one or more intestinal marker genes is detected bydetermining the presence of the gene products encoding said markergenes, wherein the gene products are selected from the group consistingof an mRNA and a protein.
 16. The method of claim 15, wherein thepresence of a protein encoding an intestinal marker gene is determinedby using a specific antibody.
 17. A kit of parts for performing themethod as defined in claim
 1. 18. The kit of claim 17 for performing themethod as defined in claim
 12. 19. A kit of parts for performing amethod for identifying a compound inhibiting the development of anintestinal phenotype in one or more cells having a predisposition todevelop an intestinal phenotype, wherein said cells are characterized bythe loss of expression of the RUNX3 gene and the expression of one ormore intestinal marker genes, the method comprising: (a) contacting saidone or more cells with a solution supposed to contain at least onecompound to be identified; (b) incubating said cells for a predeterminedperiod of time; and (c) measuring the expression of the RUNX3 gene aswell as the expression of said one or more intestinal marker genes. 20.A kit of parts for performing a method for identifying a compoundinhibiting the development of an intestinal phenotype in one or moregastric cells which are in a precancerous state, the method comprising:(a) contacting said one or more gastric cells with a solution supposedto contain at least one compound to be identified; (b) incubating saidcells for a predetermined period of time; and (c) measuring theexpression of the RUNX3 gene and the expression of said one or moreintestinal marker genes, wherein said intestinal phenotype ischaracterized by the loss of expression of the RUNX3 gene and a changein the expression of one or more intestinal marker genes such that thelevel of said one or more marker genes is characteristic of saidintestinal phenotype; wherein a compound inhibiting the development ofan intestinal phenotype of one or more gastric cells is identified.